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Cryopreservation of chicken sperm: The enigma of glycerol
ABSTRACTS,
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constitution of offspring. However, recent research indicates that preserving male gametes alone may not yield the desired results because: (1) mitochondria and other cytoplasmic inheritance factors are transmitted only from mother to offspring; and (2) functional expression of some nuclear genes is determined by whether the gene was inherited from the male or female parent (“parental imprinting”). The judicious cryopreservation and use of oocytes and/or embryos from female founders provide the only means of conserving maternally transmitted genetic variability. Live births have been reported after the transfer of cryopreserved embryos in 13 species, including 4 captive-bred nondomestic species (baboon, eland, marmoset, and macaque monkey). To date, only one species, the pig, has resisted all attempts using standard embryo cryopreservation techniques. The cryopreservation of mature mammalian oocytes has been less successful probably due to disruption of the spindle and disorientation of chromosomes during arrest in metaphase II. The practical application of embryo cryopreservation to the propagation of rare wild mammals requires: (1) a suitable number of fertile founder animals; (2) an extensive understanding of the behavior and reproductive physiology of the species for control of reproductive cycles; (3) safe anesthetic techniques for manipulative procedures; (4) systematic sampling of female founders based on sound genetic criteria; (5) a fertile population of embryo recipients of the same or taxonomically similar species; and (6) secure, long-term storage facilities. An example of the application of embryo cryopreservation and banking to an endangered antelope, the scimitar-homed oryx (Oryx dammah), will be discussed. (Research sponsored, in part, by NOAHS Center and Friends of the National Zoo.) 81. Cryopreservation
of Chicken Sperm: The Enigma of Glycerol. ROY H. HAMMERSTEDT AND JAMES K. GRAHAM (Biochemistry Program,
The Pennsylvania State University, University Park, Pennsylvania 16802). Empirical conditions have been identified that result in the completion of a freeze-thaw cycle for rooster sperm with a high retention of fertility, but the best cryoprotectant (glycerol) has a contraceptive effect in subsequent inseminations. Similar, but less extreme, contraceptive effects in human and stallion inseminations have been reported. This report will first review recent data for cryopreservation of poultry sperm, and then summarize data establishing a contraceptive effect of glycerol. Subsequent discussions will focus on the effects of molar concentrations of glycerol on: (a) physical features of the cytoplasm (cytoplasmic organization and viscosity); (b) alteration of permeability and stability of the membrane bilayer(s); and (c) noncovalent attachment of the proteins to the sperm sur-
.
ANNUAL
MEETING
563
face. Potential additional effects of glycerol on cellular metabolism will be evaluated, with the intent of emphasizing the importance of maintenance of bioenergetic balance during the temperature changes associated with any cryopreservation protocol. Limits to the successful removal of glycerol from sperm suspensions will be outlined. Finally, the potential value of several unique genetic lines of roosters (differing in their capacities to survive a freeze-thaw cycle) to analyzing these effects will be outlined. (Supported by USDA 88-01357.) 82. Status of Cryopreservation of Embryos from Domestic Animals. M. L. FAHNING AND M. DE GARCIA (University of Minnesota, St. Paul,
Minnesota; and CryovaTech Inc., Hudson, Wisconsin).
International,
The discovery of glycerol as an effective cryoprotectant for spermatozoa subsequently led to research on cryopreservation of embryos. The first successful offspring from frozen-thawed embryos were reported in the mouse, later in other laboratory animals, and subsequently, in domestic animals. Research in cryopreservation techniques have included studies concerning the type and concentration of cryoprotectant, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal. To date, successful results based on pregnancy rates have been obtained with cryopreserved cow, sheep, goat, and horse embryos with no success reported in swine. Post-thaw embryo survival has been shown to be dependent on the initial embryo quality, developmental stage, and species. The freezing techniques most frequently used in research and by commercial companies are identitied as “equilibrium” cryopreservation. In this technique the embryos are placed in a concentrated glycerol solution (1.4 M in PBS supplemented with bovine serum albumin) at room temperature and the glycerol is allowed to equilibrate for a 20-min period. As the cooling process continues, the straws are seeded ( - 4 to -7°C) and cooling is continued at a rate of 0.3 to O.S”C/min down to - 30°C when bovine embryos may be plunged into LN,. Sheep embryos are successfully frozen with ethylene glycol (1.5 M) or DSMO (1.5 M) rather than with glycerol. Horse embryos have been frozen in 0.5 rather than 0.25 cc straws but with similar cooling rates, seeding, and plunging temperature as bovine embryos. The high sensitivity of pig embryos to decreased temperatures and to treatment with cryoprotectants is thought to be due to their lipid content resulting in high percentage of degenerated embryos when cooled to 10-15°C. Recent research has shown that embryos may also be frozen by a “nonequilibrium” method. This rapid freezing by vitrification consists of dehydration of the embryo at room temperature by a very highly concentrated solu-
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constitution of offspring. However, recent research indicates that preserving male gametes alone may not yield the desired results because: (1) mitochondria and other cytoplasmic inheritance factors are transmitted only from mother to offspring; and (2) functional expression of some nuclear genes is determined by whether the gene was inherited from the male or female parent (“parental imprinting”). The judicious cryopreservation and use of oocytes and/or embryos from female founders provide the only means of conserving maternally transmitted genetic variability. Live births have been reported after the transfer of cryopreserved embryos in 13 species, including 4 captive-bred nondomestic species (baboon, eland, marmoset, and macaque monkey). To date, only one species, the pig, has resisted all attempts using standard embryo cryopreservation techniques. The cryopreservation of mature mammalian oocytes has been less successful probably due to disruption of the spindle and disorientation of chromosomes during arrest in metaphase II. The practical application of embryo cryopreservation to the propagation of rare wild mammals requires: (1) a suitable number of fertile founder animals; (2) an extensive understanding of the behavior and reproductive physiology of the species for control of reproductive cycles; (3) safe anesthetic techniques for manipulative procedures; (4) systematic sampling of female founders based on sound genetic criteria; (5) a fertile population of embryo recipients of the same or taxonomically similar species; and (6) secure, long-term storage facilities. An example of the application of embryo cryopreservation and banking to an endangered antelope, the scimitar-homed oryx (Oryx dammah), will be discussed. (Research sponsored, in part, by NOAHS Center and Friends of the National Zoo.) 81. Cryopreservation
of Chicken Sperm: The Enigma of Glycerol. ROY H. HAMMERSTEDT AND JAMES K. GRAHAM (Biochemistry Program,
The Pennsylvania State University, University Park, Pennsylvania 16802). Empirical conditions have been identified that result in the completion of a freeze-thaw cycle for rooster sperm with a high retention of fertility, but the best cryoprotectant (glycerol) has a contraceptive effect in subsequent inseminations. Similar, but less extreme, contraceptive effects in human and stallion inseminations have been reported. This report will first review recent data for cryopreservation of poultry sperm, and then summarize data establishing a contraceptive effect of glycerol. Subsequent discussions will focus on the effects of molar concentrations of glycerol on: (a) physical features of the cytoplasm (cytoplasmic organization and viscosity); (b) alteration of permeability and stability of the membrane bilayer(s); and (c) noncovalent attachment of the proteins to the sperm sur-
.
ANNUAL
MEETING
563
face. Potential additional effects of glycerol on cellular metabolism will be evaluated, with the intent of emphasizing the importance of maintenance of bioenergetic balance during the temperature changes associated with any cryopreservation protocol. Limits to the successful removal of glycerol from sperm suspensions will be outlined. Finally, the potential value of several unique genetic lines of roosters (differing in their capacities to survive a freeze-thaw cycle) to analyzing these effects will be outlined. (Supported by USDA 88-01357.) 82. Status of Cryopreservation of Embryos from Domestic Animals. M. L. FAHNING AND M. DE GARCIA (University of Minnesota, St. Paul,
Minnesota; and CryovaTech Inc., Hudson, Wisconsin).
International,
The discovery of glycerol as an effective cryoprotectant for spermatozoa subsequently led to research on cryopreservation of embryos. The first successful offspring from frozen-thawed embryos were reported in the mouse, later in other laboratory animals, and subsequently, in domestic animals. Research in cryopreservation techniques have included studies concerning the type and concentration of cryoprotectant, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal. To date, successful results based on pregnancy rates have been obtained with cryopreserved cow, sheep, goat, and horse embryos with no success reported in swine. Post-thaw embryo survival has been shown to be dependent on the initial embryo quality, developmental stage, and species. The freezing techniques most frequently used in research and by commercial companies are identitied as “equilibrium” cryopreservation. In this technique the embryos are placed in a concentrated glycerol solution (1.4 M in PBS supplemented with bovine serum albumin) at room temperature and the glycerol is allowed to equilibrate for a 20-min period. As the cooling process continues, the straws are seeded ( - 4 to -7°C) and cooling is continued at a rate of 0.3 to O.S”C/min down to - 30°C when bovine embryos may be plunged into LN,. Sheep embryos are successfully frozen with ethylene glycol (1.5 M) or DSMO (1.5 M) rather than with glycerol. Horse embryos have been frozen in 0.5 rather than 0.25 cc straws but with similar cooling rates, seeding, and plunging temperature as bovine embryos. The high sensitivity of pig embryos to decreased temperatures and to treatment with cryoprotectants is thought to be due to their lipid content resulting in high percentage of degenerated embryos when cooled to 10-15°C. Recent research has shown that embryos may also be frozen by a “nonequilibrium” method. This rapid freezing by vitrification consists of dehydration of the embryo at room temperature by a very highly concentrated solu-